Lsm image examiner software

To quantify BrdU + cells by immunohistochemistry, photomicrographs were taken using a light Olympus BX60 microscope at 20X amplification using a motorized stage. Three to four brain slices at the level of the AOB and MOB per rat were analyzed (Figures 2A,B, respectively). Brain slices were matched neuroanatomically across all animals. Eight females were evaluated per group. The MOB layers were delimited by three circles of 400 μm in diameter to quantify the number of BrdU + cells. In the AOB three 200 μm diameter circles delimitated the layers. The number of cells in each circle per layer were added and we calculated the mean values from 3 to 4 brain slices. The ImagePro program was used to automatically quantify the number of BrdU + cells in each layer.
For immunofluorescence 20X photomicrographs from four animals in each group were taken of the MOB and AOB using a confocal Zeiss LSM 700. The number of BrdU + cells and the double labeling (BrdU/NeuN+) were evaluated with the orthogonal tool of the Zeiss LSM Image Examiner software.

Cells were seeded on coverslips in 24-well plates and transfected with various expression constructs for 24–36 h. They were then stained for immunofluorescence detection using confocal fluorescence microscopy or directly visualized for cells expressing GFP or mCherry-tagged proteins as previously described^{91 (link)}. Briefly, the cells were washed with PBSCM buffer (PBS buffer supplemented with 10 mM CaCl₂ and 10 mM MgCl₂) followed by fixation with 3% paraformaldehyde in PBSCM. The fixed cells were then washed with PBSCM containing 50 mM NH₄Cl and permeabilized with PBSCM containing 0.1% saponin. For immunostaining, the antibodies were diluted in PBSCM containing 7% fetal bovine serum and 2% bovine serum albumin. Images were collected using a 63× oil immersion objective with appropriate laser excitation on a Zeiss LSM 800 confocal microscope. The detector gain was first optimized by sampling various regions of the coverslip and then fixed for each specified channel. Once set, the detector gain value was kept constant throughout the image acquisition process. Images were analyzed using Zeiss LSM Image Examiner Software.

All imaging was performed with a Zeiss LSM 510 Confocal microscope (Carl Zeiss, Toronto, ON). Live cell imaging of mitochondrial membrane potential (TMRM; Invitrogen, Burlington, ON) and mitochondrial ROS (Mitosox; Invitrogen, Burlington, ON) were imaged as previously described. Primary antibodies used include VEGF (1:100; Santa Cruz Biotechnologies), HIF-1a (1:100; Abcam, San Francisco, CA), vWF (1:200; Abcam, San Francisco, CA, San Francisco, CA), SMA (1:100; Abcam, San Francisco, CA). For imaging of hypoxic cells, stains were applied onto cells within the hypoxic incubator, and imaged within in a custom chamber suffused with 5 % oxygen gas to preserve hypoxic conditions. Lectin fluorescein ricinus communis agglutinin I (5 mg) (Vector Laboratories, Inc, Burlingame, CA) was injected via a central venous cannula for 5 min prior to sacrifice, gastrocnemius isolation and flash freezing. For semi-quantification, randomly selected fields were evaluated and regions of interest were semi-quantified in arbitrary fluorescence units using the Zeiss LSM Image Examiner software version 3.0.2.70 (Carl Zeiss, Toronto, ON). Confocal laser settings were adjusted for background signals from a negative control (secondary antibody only) would register an intensity of zero. For HIF-1α, only nuclear signals were included in the regions of interest.

To visualize γ-ray-induced γ-H2AX foci, cells cultured on coverslips were washed twice with PBS and fixed in 100% ice cold methanol for 10 min, followed by permeabilization with 0.3% Triton X-100 for 15 min at room temperature. Next, the coverslips were washed three times with PBS, followed by blocking with 0.1% bovine serum albumin in PBS for 1 h at room temperature. The cells were immunostained using primary antibody directed against the γ-H2AX proteins overnight at 4 °C. After 3 times being washed with PBS, the cells were stained with the appropriate secondary antibodies conjugated with Alexa Fluor 488- or Alexa Fluor 594 (green and red fluorescence, respectively; Molecular Probes, Eugene, OR, USA). The coverslips were mounted onto the slides using Vectashield mounting medium containing 4′, 6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA). Fluorescence images were taken under a confocal microscope (Zeiss LSM 510 Meta; Carl Zeiss, Jena, Germany) and analyzed with Zeiss LSM Image Examiner software (Carl Zeiss). Percentage was calculated among at least 100 cells by dividing the number of γ-H2AX foci-positive cells by the number of DAPI-stained cells.